1、Designation: E2180 07 (Reapproved 2017)E2180 18Standard Test Method forDetermining the Activity of Incorporated AntimicrobialAgent(s) In Polymeric or Hydrophobic Materials1This standard is issued under the fixed designation E2180; the number immediately following the designation indicates the year o
2、foriginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.INTRODUCTIONPolymeric materials such as vinyl pool liners, shower curt
3、ains, and various medical devices aretreated frequently with incorporated or bound antimicrobial agents. Practices G21 is used to determinethe ability of polymer materials to resist microbial attack or staining (see also Practice E1428);however, none of the methods permit quantitative evaluations of
4、 incorporated antimicrobial activity.2These antimicrobials typically require contact with the microbial cell for maximal activity. Whenaqueous based bacterial inoculum suspensions are applied onto a preservative-treated plastic or otherhydrophobic material, the surface tension of the polymer often c
5、auses the inocula suspension to dome.Bacteria within the drops of inoculum may not contact the treated surface if the challenged surfacedoes not dry, or upon drying, cells may become layered. This test standard involves an agar slurryinoculum vehicle that provides a relatively uniform contact of the
6、 inocula with antimicrobial-treatedhydrophobic surfaces.1. Scope1.1 This test method is designed to evaluate (quantitatively) the antimicrobial effectiveness of agents incorporated or bound intoor onto mainly flat (two dimensional) hydrophobic or polymeric surfaces. The method focuses primarily on a
7、ssessing antibacterialactivity; however, other microorganisms such as yeast and fungal conidia may be tested using this method.1.2 The vehicle for the inoculum is an agar slurry which reduces the surface tension of the saline inoculum carrier and allowsformation of a “pseudo-biofilm,” providing more
8、 even contact of the inoculum with the test surface.NOTE 1This test method facilitates the testing of hydrophobic surfaces by utilizing cells held in an agar slurry matrix. This test method, as written,is inappropriate to determine efficacy against biofilm cells, which are different both genetically
9、 and metabolically than planktonic cells used in this test.1.3 This method can confirm the presence of antimicrobial activity in plastics or hydrophobic surfaces and allows determinationof quantitative differences in antimicrobial activity between untreated plastics or polymers and those with bound
10、or incorporatedlow water-soluble antimicrobial agents. Comparisons between the numbers of survivors on preservative-treated and controlhydrophobic surfaces may also be made.1.4 The procedure also permits determination of “shelf-life” or long term durability of an antimicrobial treatment which maybe
11、achieved through testing both non-washed and washed samples over a time span.1.5 Knowledge of microbiological techniques is required for these procedures.1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.7 This standard do
12、es not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine theapplicability of regulatory limitations prior to use.1.8 This
13、 international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT
14、) Committee.1 This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2017May 1, 2018. Published April 2017May 2018.
15、 Originally approved in 2001. Last previous edition approved in 20122018 asE2180 07(2012).(2017). DOI: 10.1520/E2180-07R17.10.1520/E2180-18.2 Price, D. L., Sawant, A. D., and Ahearn, D. G., “Assessment of the antimicrobial activity of an insoluble quaternary amine complex in plastics,” J. Industr. M
16、icrobiol,Vol 8, No. 2, 1991, pp. 8389.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM r
17、ecommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States12. Referen
18、ced Documents2.1 ASTM Standards:3E1054 Test Methods for Evaluation of Inactivators of Antimicrobial AgentsE1428 Test Method for Evaluating the Performance of Antimicrobials in or on Polymeric Solids Against Staining byStreptomyce species (A Pink Stain Organism)G21 Practice for Determining Resistance
19、 of Synthetic Polymeric Materials to Fungi3. Terminology3.1 Definitions:3.1.1 agar slurry, na semi-gelatinous liquid formed when 3 g/L agar-agar is added to a 0.85 % saline solution.3.1.2 inoculum, nin microbiology, a specimen comprised of living spores, bacteria, single celled organisms, or other l
20、ivematerials, yeast or the multicellular filamentous fungi, or combination of two or more types of microorganisms, that are introducedinto a test medium or onto a specimen to be tested in order to investigate the lability of the medium or specimen to supportmicrobial growth or to investigate its ant
21、imicrobial properties.3.1.3 inoculum vehicle, nthe carrier solution used to transport bacterial cells to a test surface. Samples include saline, nutrientbroth, tryptic soy broth, agar slurry, or other buffers that maintain bacterial viability.the carrier solution used to transport theinoculum to a g
22、iven sample or object.3.1.4 neutralizing recovery broth, nliquid growth media used to inactivate the effects of the test antimicrobial agent.4. Summary of Test Method4.1 This method involves inoculation of a molten (45C)(45 C) agar slurry with a standardized culture of bacterial cells.4.2 A thin lay
23、er of the inoculated agar slurry (0.5-1.0 mL) is pipetted onto the test and untreated control material (triplicatesamples minimum).4.3 After the specified contact time (24 h commonly used), surviving microorganisms are recovered via elution of the agarslurry inoculum from the test substrate into neu
24、tralizing broth and extracted via methods that provide complete removal of theinoculum from the test article (examples include sonication, vortexing, and/or manual extraction, that is, stomacher).4.4 Serial dilutions are made, then pour or spread plates are made of each dilution.Agar plates and dilu
25、tion broths are incubatedfor 48 6 2 62 h at a specified temperature dependent upon the optimal temperature for test organism.4.5 Bacterial colonies from each dilution series are counted and recorded.4.6 Calculation of percent reduction of bacteria from treated versus untreated samples is made.5. Sig
26、nificance and Use5.1 This method can be used to evaluate effectiveness of incorporated/bound antimicrobials in hydrophobic materials such asplastics, epoxy resins, as well as other hard surfaces.5.2 The aqueous based bacterial inoculum remains in close, uniform contact in a “pseudo-biofilm” state wi
27、th the treatedmaterial. The percent reduction in the surviving populations of challenge bacterial cells at 24 h versus those recovered from anon-treated control is determined.5.3 The hydrophobic substrate may be repeatedly tested over time for assessment of persistent antimicrobial activity.6. Appar
28、atus6.1 Erlenmeyer Flask, 250 mL.6.2 Petri Dishes, (15 100 mm), sterile.6.3 Colony Counter.6.4 Specimen Cups, (120 mL), sterile or equivalent sterile equipment for extraction.6.5 Pipetters, (1000 L) positive displacement.6.6 Pipette Tips, sterile.6.7 Test Tubes, 16 100 mm.6.8 Incubator, set at requi
29、red temperature (25-35 6 2C).2 C).6.9 Autoclave.3 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.E2180 1826.10
30、 Water Bath, capable of maintaining water at 45 6 2C.2 C.6.11 Sterile Cotton Swabs.6.12 Sonic Bath, 47 Khz, cleaning non-cavitating.6.13 Vortex Mixer.6.14 pH Meter.6.15 Hot Plate, with stirrer.6.16 Spectrophotometer, set at 600 nm.6.17 Sterile Cuvettes.6.18 Test Materials, sterile if specified by in
31、terested parties.6.19 Cell Counting Chamber.7. Reagents7.1 Media:7.1.1 Tryptic Soy Broth, or appropriate broth.7.1.2 Tryptic Soy Agar, or appropriate agar.7.1.3 Neutralizing Broth, appropriate for the antimicrobial compound tested.(See Practice E1054.)7.1.4 Agar-agar.7.1.5 NaCl.7.1.6 Sterile Deioniz
32、ed Water.7.1.7 Sterile 0.85 % Saline Dilution Blanks, 9.0 mL in 16 100 mm test tubes or appropriate dilution buffer (such as phosphatebuffer or Butterfields buffer).7.2 Test OrganismsSpecific organisms are recommended but choice of organism should be relevant to the environment inwhich the product w
33、ill perform.7.2.1 Gram-positive bacteria Staphylococcus aureus ATCC 6538.7.2.2 Gram-negative bacteria Pseudomonas aeruginosa ATCC 15442 or Klebsiella pneumoniae ATCC 4352.7.2.3 Other microorganisms such as yeast or fungal conidia may also be tested using this procedure. Exposure periods may bemodifi
34、ed (up to 96 h) to address more resistant microorganisms.8. Procedure8.1 Grow 18 h bacterial cultures (three transfers) at a specified temperature dependent upon the optimal temperature for the testorganism in tryptic soy or appropriate broth. These cultures should originate from 18-24 h growth comi
35、ng from stock culture platesor growth on agar slants.8.2 Prepare the agar slurry by dissolving 0.85 g NaCl and 0.3 g agar-agar in 100 mL of deionized water. Heat with stirring ona hot plate until the agar dissolves. One agar slurry should be prepared for each organism tested.8.3 Sterilize the agar s
36、lurry by autoclaving for 15 min at 121 6 2C,2 C, 15 psi., then equilibrate at 45 6 2C.2 C.8.4 Prepare 3.0 3.0 cm square samples of the treated and control test materials (sample minimum of triplicates for “0” hcontrols, incubation period treated samples and incubation period control samples for each
37、 test organism).8.5 Place each sample into a sterile 15 100 mm petri dish.8.6 Adjust bacterial broth cultures to 1-5108 cells/mL with a spectrophotometer or cell counting chamber.8.7 Dip a cotton swab into sterile 0.85 % saline (with or without a non-inhibitory levels of a surfactant) and pre-wet th
38、e testsample. This will aid in dispersing the agar slurry evenly on the sample.8.8 Place 1.0 mL of standardized culture (1-5108 cells/mL) into the 100 mL agar slurry equilibrated at 45 6 2C.2 C. Thefinal concentration should be 1-5106 cells/mL in the molten agar slurry.8.9 Pipet 0.5-1.0 mL of inocul
39、ated agar slurry onto the test and control samples. Slow, gentle application at a low angle ofincidence relative to the sample will aid in formation of a film no more than 1 mm in depth. If the inoculum volume and/or surfacearea of the sample are modified, the inoculum volume should be adjusted to p
40、rovide an agar slurry 1 mm in depth over the entiresample surface. The inoculum volume and surface area tested should be reported.NOTE 2For substrates found to be extremely hydrophobic, addition of non-inhibitory levels of a surfactant to the agar slurry may also aid in surfacewetting properties. Al
41、ternatively, the slurry may be cooled to 25 6 2C2 C then applied to the polymer surface. Application of pre-cooled agar slurryhas also been shown to aid in the dispersion of the inoculum over an extremely hydrophobic test surface.8.10 Allow the agar slurry inoculum to gel and then place the samples
42、in an incubator at a specified temperature dependent uponthe optimal temperature for the test organism or one which mimics the temperature in which the test substrate will be utilized forE2180 183the specified exposure period (usually 24 6 2 h). Low humidity in the incubator can cause drying of the
43、agar slurry inoculum onthe samples, therefore relative humidity within the incubator should be at or above 75 %. This can be accomplished with openreservoirs of certain saturated salt in water solutions.48.11 Make serial dilutions (as described in 8.12 8.16) of the agar slurry recovered immediately
44、from “0” h control samplesand spread or pour plate each dilution to determine cfu/mL recoverable at time “0 h.”8.12 Following the specified contact time, aseptically remove the incubation period control samples and incubation periodtreated samples from the petri dishes to 120 mL specimen cups or oth
45、er suitable container containing a sufficient volume ofneutralizing broth to form an initial 1:10 or 1:100 dilution of the original inoculum.8.13 Place the specimen cups containing the recovered test samples into a non-cavitating sonic bath and sonicate for 1 min.8.14 Sonication should be followed b
46、y 1 min of vigorous mechanical vortexing. This should facilitate the complete release ofthe agar slurry from the sample. Note that the test surface may be imprint cultured onto tryptic soy agar following sonication andvortexing in order to determine release efficiency of the inoculum from the treate
47、d surface.8.15 Perform serial dilutions of the initial neutralizing broth sufficient to include one dilution beyond the original inoculum (asdetermined in 8.11).8.16 Spread or pour plate each dilution into tryptic soy agar or other appropriate agar (for example, Sabourauds agar for fungi)and incubat
48、e plates at an optimal temperature for the test organism for 48 6 2 h.8.17 Count and record colony numbers for each dilution plate.9. Calculation9.1 Determine the geometric mean of the number of organisms recovered from the triplicate incubation period control andincubation period treated samples by
49、 the following equation:geometric mean5Log10X11Log10X21Log10X3!3 (1)where:X = number of organisms recovered from the incubation period control or incubation period treated samples.9.2 Percent ReductionUse the following equation to calculate the percent reduction:%reduction5a 2b!3100a (2)where:a = the antilog of the geometric mean of organisms recovered from the incubation period control samples (as determined in 9.1),andb = the antilog of the geometric mean of the number of organisms recovered from the incubation period treated samples (
